Figure 1. Design and characterisation of an analog sensitive allele of C elegans
pkc-3:(A) Structure and homology of the PKC-3 active site. Predicted structure of the C. elegans PKC-3 kinase domain (AlphaFold) showing gatekeeper residue (blue). Superposition of the bound ATP analogue (green) obtained by structural superposition with PKC beta (pdb code 3PFQ) as in Hannaford et al. (2019). Amino acids around the position of the gatekeeper site (blue) and the suppressor site (yellow) appear well conserved. (B, C) The analog sensitive PKC-3, PKC-3AS, excludes PAR-2 from the anterior as efficiently as wild-type. Example midsection confocal images shown along with quantification of PAR-2 membrane concentration profiles and PAR-2 domain size for pkc-3AS (n=11) and wild-type control embryos (n=12). (D) Inhibition of PKC-3AS by the ATP analogue 1NA-PP1 results in dose-dependent loss of PAR-2 asymmetry. Example midsection confocal images of control or pkc-3AS embryos expressing GFP::PAR-2 treated with the indicated concentration of 1NA-PP1. Quantitation of asymmetry index shown at right. Sample sizes: control - 0 uM (n=6), 1 uM (n=6), 5 uM (n=5), 10 uM (n=4), 20 uM (n=5), 100 uM (n=5); pkc-3AS - 0 uM (n=5), 1 uM (n=5), 5 uM (n=6), 10 uM (n=6), 20 uM (n=6), 100 uM (n=5). IC50 was calculated by bootstrapping to obtain a mean and a 95% confidence interval, mean = 7.18, bounds = 6.03, 8.42. (E, F) Inhibition of PKC-3AS is as efficient or better at reducing PAR-2 asymmetry compared to established methods. For comparison, asymmetry index of PAR-2 is shown for the temperature sensitive
pkc-3(
ne4246) allele at the restrictive temperature (control, n=7;
pkc-3(ts), n=9) (E) and after RNAi targeting
pkc-3 (control(RNAi), n=7;
pkc-3(RNAi), n=10) (F). (G) Inhibition of PKC-3AS is rapid. A timeseries of midsection confocal images of either control or pkc-3AS embryos expressing GFP::PAR-2 treated with 1NA-PP1 or DMSO (control). Open arrowheads (white) indicate ectopic GFP::PAR-2 localisation at the anterior pole, which begins to appear by one minute after wash-in of 1NA-PP1. (H) Inhibition of PKC-3AS is reversible. GFP::PAR-2-expressing embryos were treated with 1NA-PP1 (50uM) at the one-cell stage to prevent polarization. Embryos were allowed to divide symmetrically and then subject to buffer exchange in the presence or absence of 1NA-PP1 immediately following cytokinesis. Reactivation is evident by the formation of a polarized PAR-2 cap (closed arrowhead, yellow) and clearance of PAR-2 in the anterior (open arrowhead, yellow). Scale bars, 20μm. Individual data points or mean ± SD shown for all plots.